PROJECT SUMMARY A major obstacle in the development of mold-active antifungals is limited methodologies for high throughput screening with molds. Aspergillus fumigatus and Fusarium spp. are two common molds that cause invasive infections in immune-compromised patients. Despite current antifungal treatments, limited to only three unique classes of drugs, mortality rates of these infections are devastatingly high. In addition to the serious health threat molds pose to immune-compromised patients, molds cause infections in immune-competent individuals, particularly in the form of corneal infections, or fungal keratitis. Fungal keratitis is a leading cause of monocular blindness worldwide and only about half of patients with corneal infections caused by Aspergillus or Fusarium respond to treatment. The current state of fungal infections worldwide, combined with emerging resistance to current antifungal drugs, calls for novel classes of mold-active antifungals to combat these life- threatening infections. This proposal aims to fill a technical gap in the field by applying the adenylate kinase assay to filamentous fungi to perform high throughput screening. The adenylate kinase assay has been validated and successfully used for high throughput screening with yeast and bacteria and possesses several qualities that make it an excellent candidate for application to molds. We have shown that this assay reliably detects lysis of A. fumigatus and optimized conditions suitable for high throughput screening. In this proposal, our goal is to adapt this assay to Fusarium oxysporum, then perform screens with Aspergillus and Fusarium with multiple small molecule and natural product libraries. In addition to screening against wild type A. fumigatus, we will perform a repurposing screen of FDA-approved drugs using a strain harboring a common voriconazole resistance mechanism. In this way, we hope to identify novel classes of mold-active antifungal drugs, as well as drugs to be repurposed and expedited to clinical use to treat fungal infections.